CN1280490A - Peptide/lipid complex formation by co-lyophilization - Google Patents

Peptide/lipid complex formation by co-lyophilization Download PDF

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CN1280490A
CN1280490A CN98811818A CN98811818A CN1280490A CN 1280490 A CN1280490 A CN 1280490A CN 98811818 A CN98811818 A CN 98811818A CN 98811818 A CN98811818 A CN 98811818A CN 1280490 A CN1280490 A CN 1280490A
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peptide
lipid
palmityl
phosphatidylcholine
complex formation
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CN100415210C (en
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琼-路易斯·达索克斯
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JEAN LOUIS DASSEUX
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1275—Lipoproteins; Chylomicrons; Artificial HDL, LDL, VLDL, protein-free species thereof; Precursors thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers

Abstract

The invention relates to the formation of peptide/lipid vesicles and complexes through the co-lyophilization of peptides, preferably that are able to adopt an amphipathic alphahelical conformation, and one or more lipids. A single solution which solubilizes both the peptides and lipids or two separate solutions may be lyophilized.

Description

Freeze-drying prepares peptide/peptide/lipid complex formation altogether
1. invention field
The present invention relates to peptide and one or more lipids, prepare peptide/lipid microcapsule and complex by being total to freeze-drying, preferred use can be taked the peptide of both sexes alpha-helical conformation.Can the two same solution or two kinds of other solution lyophilisation of branch of peptide and lipid will can be dissolved.This method is used to prepare stable peptide/lipid microcapsule and complex, comprises a large amount of and is suitable for the microcapsule of the smaller dose unit of dosage, spherical and discoid complex, but be not limited to this.
2. background technology
Liposome comprises the microcapsule that a moisture nuclear is formed by one deck lipid bimolecular film at least.Usually, phospholipid contains the lipid bilayer, but bilayer can contain other lipid.Aqueous solution in the liposome is called the absorption volume.
Liposome has developed into the microcapsule that transmits medicine, cosmetics, is used for the bioactive compound of others.The lipid bilayer is bundled into capsule with medicine, cosmetics, bioactive compound etc., and in the absorption volume of liposome, when the lipid bilayer contacted with cell surface membrane, medicine was from the discharging of liposome nuclear.Liposome is discharged into content in the cell by lipid exchange, fusion, cytophagy or absorption.See Ostro etc., 1989, the record of United States Hospital pharmaceutical journal 46:1576.On the other hand, can or be bumped in the lipid bimolecular film of microcapsule combinations such as medicine, cosmetics, bioactive compounds.
Except that microcapsule, with the graininess complex transportation of substances that contains lipid.As, many research worker have found that it can be used for preparing lipoprotein granule or the complex that the reconstruct of similar volume and density is arranged to high density lipoprotein (HDL) granule.These reconstruct complex contain apoprotein (normally apo A-1) and the phospholipid such as the lecithin of purification usually.Sometimes, also comprise nonesterified cholesterol.Preparing the most frequently used method of these granules is (1) carries out component by water-bath sonication or probe sonication supersound process, (2) spontaneously interact between protein component and the preformed lipid granule, (3) by the reconstruct of detergent mediation, remove detergent by dialysis then.Jonas, 1986, Enzymology method, 128:553-582; Lins etc., 1993, biochemistry and biophysics's journal, 1151:137-142; Brouillette﹠Anantharamaiah, 1995 biochemistrys and biophysics's journal, 1256:103-129; Jonas, 1992, the structure of apoprotein and function, the 8th chapter: 217-250.Also form similar complex by replacing the apoprotein component with both sexes screw type peptide.Insufficient is that the whole bag of tricks all exists bigger problem for form a large amount of pure complex on rational cost-usefulness basis.And, the peptide of the unexposed both sexes alpha-helical conformation of these publications/or the analog of peptide and lipid cryodesiccated method altogether.
Some technology of preparation lipid microcapsule and complex are known.Prepare microcapsule with various schemes, or liposome, thereby dissimilar microcapsules formed.Various types of liposomees comprise: multilamellar microcapsule, little monolayer microcapsule and big monolayer microcapsule.
The hydration of carrying out phospholipid (or other lipid) with aqueous solution also can produce lipid decentralized photo and spontaneous formation multilamellar microcapsule (" MLVs ").A kind of MLV has multilamellar lipid bilayer to surround the liposome of central moisture nuclear.The liposome of these types is bigger than little monolayer microcapsule (SUVs), and its diameter is 350-400nm.In a round-bottomed flask, lipid is dissolved in the chloroform, and evaporation removes chloroform and forms skim until lipid on flask walls, thus preparation MLVs.Add aqueous solution, make lipid layer rehydrated.Rotary flask forms microcapsule.Deamer etc., 1983, liposome (Ostro, Ed.), Marcel Dekker, Inc. New York (quote Bangham etc., 1965, molecular biology magazine 13:238).Johnson etc. report that subsequently the method also can produce the monolayer microcapsule, see Johnson etc., 1971, and biochemistry and biophysics's journal 233:820.
Little monolayer microcapsule (SUV) is the liposome of a moisture nuclear of a kind of single lipid bilayer enclose.According to the distinct methods of preparation SUVs, the scope of its diameter is 25-110nm.The preparation method of SUVs the earliest is: the dry prepared product of the phospholipid under the inflated with nitrogen condition in chloroform adds water makes lipid concentration in the millimole scope, at 45 ℃ solution is carried out supersound process and makes it clear and bright.Deamer etc., 1983, liposome (Ostro, Ed.), Marcel Dekker, Inc. New York.Prepare SUVs with this kind mode, produce the liposome of diameter at 25-50nm.
The another kind of method for preparing SUVs be with a kind of ethanol/lipid solution inject fast a kind of will be by the aqueous solution of enclose.Deamer etc., 1983, liposome (Ostro, Ed.), MarcelDekker, Inc. New York (quote Batzri etc., 1973, biochemistry and biophysics's journal 298:1015).The SUVs diameter for preparing with this kind mode is 25-50nm.
Also can make the multilamellar microcapsule repeat by a pressure is 20,4 preparations of the French press of 000psi. SUVs.The diameter of the SUVs that makes is 30-50nm.Deamer etc., 1983, liposome (Ostro, Ed.), Marcel Dekker, Inc. New York (quote Barenholz etc., 1979, the biochemical meeting in the Europe communication 99:210 of community).
Also can prepare multilamellar and monolayer phospholipid microcapsule (Hope etc., 1996, lipid chemistry and pharmaceutics 40:89-107) by microporous membrane by the moisture prepared product that under high pressure pushes phospholipid.
Big monolayer microcapsule (LUV) is similar to the SUVs part and is, they all are the moisture nuclear that single lipid bilayer surrounds central authorities, but LUVs is bigger than SUVs.Different according to its ingredient and preparation method, the diameter of LUVs is 50-1000nm.Deamer etc., 1983, liposome (Ostro, Ed.), Marcel Dekker, Inc. New York.Usually with one of 3 kinds of methods preparation LUVs: detergent dilution method, phase inversion evaporation and injection method.
In the detergent dilution technology, use as detergent solutions such as cholate, deoxycholate, octyl group glycoside, heptyl glycoside and Triton X-100 to make lipid formulation form micelle.Then detergent is removed in the solution dialysis, formed liposome.Deamer etc., 1983, liposome (Ostro, Ed.), Marcel Dekker, Inc. New York.The method is consuming time, and can not remove detergent fully usually.The change that detergent can cause the physicochemical properties of some toxicity of Liposomal formulation and/or Liposomal formulation appears in final products.
The phase inversion evaporation technique is dissolved in lipid in water-non-polar solution, forms the micelle of phase inversion.With the non-polar solven evaporation, micelle is assembled formation LUVs.The method needs a large amount of lipid usually.
Injection method injects a kind of lipid that is dissolved in the non-polar solution will be by the aqueous solution of enclose.When non-polar solution evaporated, lipid accumulated in the solution-air interface.When gas passed through from aqueous solution, the lipid thin layer formed LUVs and the few liposome of the number of plies.Filter the size of measuring liposome.Deamer etc., 1983, liposome (Ostro, Ed.), Marcel Dekker, Deamer etc. (is quoted, 1976 in Inc. New York, biochemistry and biophysics's journal 443:629 and Schieren etc., 1978, biochemistry and biophysics's journal 542:137).Injecting preparation method needs quite high temperature when injecting, and enclose efficient may be lower.Deamer etc., 1983, liposome (Ostro, Ed.), Marcel Dekker, Inc. New York.
A target of liposome research is that development liposome preparation technology makes its long-term storage before use.As, the U.S. Patent No. 4,229,360 of Schneider etc., disclose and a kind ofly made the liposome dehydration method, and preferably made solution dehydrates with lyophilization by adding hydrophilic compounds in the colloid decentralized photo that in liquid, aqueous, forms to liposome.The example of hydrophilic compounds is low-molecular-weight chemical compounds such as high-molecular weight hydrophilic polymer or sucrose.
The U.S. Patent No. 4,411,894 of Shrank etc. discloses high concentration sucrose in the application in the Liposomal formulation of ultrasonic Treatment.Liposome contains fat-soluble product in absorbing volume, although can be with the preparation lyophilisation, even the sucrose of high concentration is arranged, this method can not stop losing of a large amount of absorption inclusions.
The U.S. Patent No. 4,857,319 of Crowe etc. discloses the purposes of disaccharidase such as sucrose, maltose, lactose and trehalose stabilized liposome in the liposome Freeze Drying Technique.The ratio of lipid content in disaccharidase and the component (w/w) is 0.1: 1 to 4: 1.Crowe adopts this method to keep the integrity of liposome, has obtained the bigger success of U.S. Patent No. 4,411,894 disclosed methods than above-mentioned Shrank.
The U.S. Patent No. 4 of Janoff etc.; 880; 635 disclose a kind of liposome dehydration method that makes, promptly under the condition that the sugar that protectiveness is arranged such as trehalose and sucrose exist with liposome vacuum lyophilization, preferably on the inside and outside layer group of lipid bilayer, all introduce the sugar of protectiveness.The method of Janoff etc. has kept competent water, so the liposome that the rehydrated back of dried lipid article produces has significant structural intergrity.
Yet the present technique field needs a kind of simple but effective method, can prepare the peptide/peptide/lipid complex formation of vacuum lyophilization, and make it rehydrated.Peptide/lipid mixtures that method of the present invention produces is stable vacuum lyophilization powder, can be directly with powder type or rehydrated after be used to prepare peptide/peptide/lipid complex formation.
3. summary of the invention
The present invention is that a kind of preparation has and the peptide of high density lipoprotein (HDL) similar characteristic or the method for protein-(phosphorus) peptide/lipid complex formation or microcapsule.This method is used a kind of solvent system, and wherein at least a peptide is dissolved in a kind of solution, and at least a lipid is dissolved in the another kind of solution.Select two kinds to be easy to miscible solution mutually.Then solution is mixed, with the lyophilization of gained solution for vacuum.
This method also can be used the second kind solvent system, wherein contains a kind of solution protein or peptide and lipid all can be dissolved in wherein.This solution can be single solution, maybe can be before adding peptide and lipid two or more solution to be mixed a kind of composite solution that forms.Peptide and lipid are dissolved in solution or the composite solution, then with peptide/lipid solution vacuum lyophilization.
The present invention preferably can take the peptide of both sexes helical configuration.In one particular embodiment of the present invention, peptide is the protein of contaminated with lipid.In another embodiment, use the replacement lipid such as analog ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, Apo E, other apolipoprotein analogues of peptide or unite use with lipid.In another specific embodiment, be similar to ApoAl analog/(phosphorus) peptide/lipid complex formation of HDL with the method preparation.The ApoAl/ peptide/lipid complex formation is used for the treatment of effectively and is not limited only to dyslipoproteinemia diseases associated-comprise that hypercholesterolemia, low HDL and aPoA-I lack, septicemia.This complex also as a token of thing be used for HDL crowd's in-vitro diagnosis, and be used for imaging technique.
Method of the present invention makes peptide/peptide/lipid complex formation preparation can parenteral, comprises animal or human's intravenous, intraperitoneal, subcutaneous, intramuscular injection and as the bolus intravenous injection of diagnosis usefulness, but is not limited to this.Further, also peptide/peptide/lipid complex formation can be mixed be used for that the human or animal is oral, the dosage form of rectum, mucosa (for example oral cavity) or local application, or be used for experiment in vitro.
This method also can be used for large-scale production both sexes peptide/phospholipid complex, contaminated with lipid protein/phospholipid complex, and/or ApoAl peptide analogues/phospholipid complex.The raw material that can prepare vacuum lyophilization is used for the preparation of large volume, or the alternative before vacuum lyophilization, blended peptide/lipid solution is placed little container (as single dosage unit), the packing unit that these are less is prepared into aseptic single dose dosage form.
With the vacuum lyophilization powder of method of the present invention preparation can be before injection rehydrated one-tenth do not have the sterile solution of microgranule, or the vacuum lyophilization powder is made the direct administration of suitable solid dosage forms.
The storage of instability or undissolved chemical compound when this method also is suitable for lacking lipid.
This method can be used to prepare the product of treatment or prevention human diseases, comprise being used for that vaccine is antigenic presents, treats or prevent dyslipoproteinemia-lack including, but not limited to hypercholesterolemia, hypertriglyceridemia, low HDL and aPoA-I jointly, and cardiovascular disease, septicemia or infectious disease such as arteriosclerosis.
The method can also prepare the complex as pharmaceutical carrier, as the vehicle (to transmit medicine, DNA, gene) of the transmission of cell outside liver or liver medicine, or as the cleanser of catching toxin (as insecticide, LPS etc.).
3.1. definition
In this application, " solvent system " refers to dissolve one or more solvent of peptide and/or lipid, if more than a kind of, is easy to miscible each other.
" peptide/peptide/lipid complex formation " refers to what lipid part and peptide formed, the particulate set of volume in high density lipoprotein (HDLs) scope.
" altogether freeze-drying " refer in the solution multiple chemical compound (as, peptide, protein, lipid, phospholipid) lyophilisation, lyophilizing or vacuum drying in same container.As, lipid solution can be mixed with the peptide solution in the same container, and with the combination solution vacuum lyophilization together that is produced, this moment, peptide and lipid were frozen drying simultaneously.
" both sexes peptide " or " both sexes alpha-helix peptide " refers to take the peptide of both sexes or both sexes helical configuration respectively.The both sexes alpha-helix is a kind of secondary structure primitive that runs in be everlasting biologically active peptide and protein.See protein: structure function and the contained Jere P.Segrest of generation origin 8:103-117 (1990), Hans de Loof, Jan G.Dohlman, the both sexes spiral type that Christie G.Brouillette and G.M.Anantharamaiah are write: classification and character.The both sexes alpha-helix is a kind of alpha-helix that opposite polarity and apolar surfaces are arranged that has with the major axis rising of spiral.Charged residue obviously distributes along pole-face.Both sexes spiral and the polar-nonpolar interface complementation of hydration phospholipid in a large number; Infer that domain that these and lipid link partly gets involved the acyl chain of fat and the interface between the terminal polar group and phospholipid and react to each other.See Jere P.Segrest. Europe biochemical meeting community communication 1976,69 (1): 111-114.
" peptide " and " protein " can be replaced mutually at this.In addition, peptide analogues of the present invention can be peptide, protein or non-peptide, promptly intends peptide.Yet the preferred bioactive molecule of all these analog.
" lipid " includes but not limited to natural and synthetic phospholipid.And then term " lipid " and " phospholipid " can exchange use at this.
4. to brief description of the drawings
Fig. 1: Superose 6 chromatographic curves of the HDL that makes from 200 μ l human serums with the density supercentrifugation.
Fig. 2 (bottom): in 1: 1 (w: (DPPC: (PVLDLFRELLNELLEALKQKLK peptide 1) of ratio preparation w); SEQ ID NO:1) Superose 6 chromatographic curves of complex.
Fig. 2 (top): in 2: 1 (w: (DPPC: Superose 6 chromatographic curves of complex peptide 1) of ratio preparation w).
Fig. 3 (bottom): in 3: 1 (w: (DPPC: Superose 6 chromatographic curves of complex peptide 1) of ratio preparation w).
Fig. 3 (top): in 4: 1 (w: (DPPC: Superose 6 chromatographic curves of complex peptide 1) of ratio preparation w).
Fig. 4 (bottom): in 5: 1 (w: (DPPC: Superose 6 chromatographic curves of complex peptide 1) of ratio preparation w).
Fig. 4 (top): in 7.5: 1 (w: (DPPC: Superose 6 chromatographic curves of complex peptide 1) of ratio preparation w).
Fig. 5: in 10: 1 (w: (DPPC: the Superose6 chromatographic curve of complex peptide 1) of ratio preparation w).
Fig. 6: at Ri=3: 1 14Superose 6 chromatographic curves of peptide 1 complex of C-labelling.
Fig. 7: at Ri=4: 1 14Superose 6 chromatographic curves of peptide 1 complex of C-labelling.
Fig. 8: at Ri=5: 1 14Superose 6 chromatographic curves of peptide 1 complex of C-labelling.
5. to detailed description of the preferred embodiments
Can synthesize or prepare both sexes alpha-helix peptide or the protein that this invention is used, contaminated with lipid protein, ApoA-I agonist peptide (agonist peptide), apoprotein analog etc. with any prior art.Peptide vacuum lyophilization preparation can be had stabilization formulations than the peptide of long shelf life, or prepare a large amount of products for preparing again, perhaps be prepared into can be before administration rehydrated and reconstitute the independent part or the dosage unit of moisture form with sterilized water or suitable buffer solution.
Known to the inventor, the present invention proposes a kind of method first, with both sexes alpha-helix peptide or peptide analogues and lipid lyophilization altogether, to form a kind of mixture that can reconstitute aseptic peptide/peptide/lipid complex formation.
In certain embodiments, can preferably will comprise and the ApoA-I analog that is not limited to ApoA-I agonist is formulated in administration in peptide-peptide/lipid complex formation.Because at medicine circulation appoplexy involving the collateral compound should be arranged the longer half-life, volume and the density when complex is similar to the HDL proteinoid especially, especially before-during β HDL protein, the half-life should prolong, so the method has some advantages.Can be by volume, feature such as density and electrophoretic mobility is divided into some groups with HDL lipid protein.By volume the more tactic examples of Zeng Jiaing are, diameter is before the micellelike of 50-60 dust-β HDL, discoid HDL with intermediate volume, i.e. the agglomerate of 65kDa (about 70 dusts), and diameter is the spherical HDL of 90-120 dust 3Or HDL 2(J.Kane, 1996 in V.Fuster, R.Ross and E.Topol[eds.] 99 pages of arteriosclerosis and coronary artery diseases; A.Tall and J.Breslow, ibid., 106 pages; Barrans etc., biochemistry and biophysics's journal 1300,73-85 page or leaf; With Fielding etc., 1995, lipid research magazine 36,211-228 page or leaf).Yet the present invention has also made the littler or bigger peptide/peptide/lipid complex formation of volume ratio HDL.
Usually can adopt following freeze-drying altogether peptide-peptide/lipid complex formation of the present invention to be prepared into stabilization formulations with long storage life.Can be used for medicine with the medicine material that freeze dried peptide-peptide/lipid complex formation prepares the large volume amount and prepare again, perhaps be prepared into can be before administration rehydrated and reconstitute the independent part or the dosage unit of moisture form with sterilized water or suitable buffer solution.
The application provides peptide that a kind of preparation has the feature that is similar to HDL or the straightforward procedure of protein-(phosphorus) peptide/lipid complex formation.Can prepare ApoA-I peptide-peptide/lipid complex formation with the method, it has the following advantages: (1) uses the most of or whole components that comprised to form the complex of estimating, has therefore avoided the waste of ubiquitous initial feed in other method.(2) be formed on freeze dried chemical compound very stable in the storage process.The complex that is produced direct hydration before use reconstitutes moisture form.(3) complex that is produced does not need further purification usually before making the back or using.(4) avoided use to comprise toxic chemicals such as detergent such as cholate.And then, can easily enlarge preparative-scale and meet GMP production requirement (promptly not having in the endotoxic environment) a kind of.
Consistent with method for optimizing is the combination in making the dissolved solvent system of the equal energy of every kind of composition of peptide and lipid.In this step, careful ground selective solvent to common dissolving with assurance both sexes peptide and hydrophilic lipid.In one embodiment, the protein or the peptide that desire can be added in the granule is dissolved in (solvent 1) in aqueous or organic solvent or the mixed solvent.(phosphorus) lipidic component is dissolved in easily and in solvent 1 blended aqueous or organic solvent or the mixed solvent (solvent 2), two solution is mixed.Perhaps (phosphorus) lipid component directly is dissolved in peptide (protein) solution.Perhaps peptide and lipid can be added the cosolvent system, in the promptly easily miscible solvent mixture.According to peptide or proteinic contaminated with lipid character, person of skill in the art will appreciate that before vacuum lyophilization solubilising or dissolving fully (and/or promote and mix) may be essential; Therefore, selective solvent correspondingly.
At first rule of thumb determine peptide (protein) and lipid proper proportion, proper physical and chemical property-this usually but always do not mean and HDL so that the complex that obtains has 2Or HDL 3Similar volume.The mol ratio of lipid and proteins/peptides should be in the scope of 2-200, according to the preferred 5-50 of the kind of required complex.Peptide/the lipid of this type of volume or protein/peptide/lipid complex formation comprise that micelle or discoid granule are (usually than HDL 2Or HDL 3Little), volume is similar to HDL 2Or HDL 3Spherical particle and compare HDL 2Bigger complex, but be not limited to this.The HDLs that we in chromatograph (Fig. 1) is used as reference material mainly is globular ripe HDL 2Before-β 1HDL is the micelle complex of the phospholipid of apolipoprotein and small number of molecules.Before-β 2HDL is the discoid complex of apolipoprotein and polymolecular phospholipid.Bonded lipid in complex (triglyceride, cholesterol, phospholipid) is many more, and the volume of HDL is big more, and its shape changes.(preceding-β 1HDL (micelle complex)  β 2HDL (discoid complex)  HDL 3(spherical complex)  HDL 2(spherical complex)).
After selecting solvent and having added peptide and lipid, freezing and vacuum lyophilization is to doing with the mixture that produces.Sometimes, in mixture, add another solvent to help vacuum lyophilization.The lyophilization product that makes can be preserved for a long time and keep stable.
In the following embodiments, peptide 1 PVLDLFRELLNELLEALKQKLK (SEQ ID NO:1) and (phosphorus) lipid are dissolved in respectively in the methanol, mix, and mix with dimethylbenzene before vacuum lyophilization then.Peptide and lipid all can be joined in the mixture of two kinds of solvents.Perhaps the methanol solution with peptide mixes with the xylene solution of lipid.Be careful and avoid peptide to saltout separating out.The peptide that containing of gained is dissolved in methanol/dimethylbenzene altogether and the solution for vacuum lyophilization of lipid form powder.
Can reconstitute solution or the suspension that moisture form obtains peptide-peptide/lipid complex formation with lyophilisation product is rehydrated.For this reason, with aqueous solution with the lyophilization powder rehydrated to a proper volume (often for ease of intravenous 5mg peptide/ml).In a preferred embodiment, with phosphate buffer or normal saline solution that the lyophilization powder is rehydrated.Mixture may be stirred or turn, rehydrated to promote, in many cases, the temperature of rehydrated reconstruction step will be equal to or greater than the phase transformation temperature (Tm) of lipidic component in the complex.In several minutes, form the solution (when complex hour forms a kind of clear solutions) of the lipid-protein complex of hydration reconstruct.
The rehydrated reconstruct preparation that can identify generation has the volume distributed median of expectation to confirm the complex in the preparation, as the volume distributed median of HDL.Can use gel filtration chromatography this moment.In the following embodiments, use Pharmacia Superose 6 FPLC gel filtration chromatography systems.Used eluent is the deionized water that contains 150mM sodium chloride.Typical sample volume is the 20-200 milliliter complex that contains 5mg peptide/ml.The flow velocity of post is 0.5ml/min.Use the protein and the people HDL of a series of known molecular amounts and Stokes diameter to calibrate chromatographic column as reference material.With 254 or light absorption or the chromatic dispersion monitor protein and the lipoprotein complexes of 280nm wavelength.
The operable solvent of the method according to this invention be nonpolar, polarity, inertia, non-inert organic solvents like ethanol, methanol, cyclohexane extraction, 1-butanols, isopropyl alcohol, dimethylbenzene, THF, ether, dichloromethane, benzene and chloroform, but be not limited to this.The present invention also uses mixed solvent except that using single solvent.And, before being used for the present invention, dry the removing of organic solvent can be anhydrated; Yet some lipid, peptide or protein can use hydration solvent or water.Changing a kind of saying is exactly, and water may be The suitable solvent, maybe can use the solvent or the organic solvent/aqueous mixtures of hydration, yet if make water, wherein must not contain detergent.As mentioned above, other solvent of preferably the purest level (to avoid concentrating of lyophilization rear impurity) should be salt-free in the solvent and do not contain granule.Yet, do not require that solvent is aseptic, because before lyophilization, after the process neutralization, can product be sterilized by the known method of pharmaceutical field, as Remington ' s materia medica, the 16th edition and 18 editions, Mack publishing company, Easton, Pennsylvania (1980 and 1990) and the described method of united states drug handbook/state-promulgated pharmacopoeia (USP/NF) X VII all are introduced in this as a reference.
The operable lipid of method for preparing the present composition comprises natural and synthetic lipid and phospholipid; comprise lower alkyl chains phospholipid; the lecithin phatidylcholine; the fabaceous lecithin phatidylcholine; two palmityl phosphatidylcholines; L-Dimyristoylphosphatidylcholine; DSPC; 1-myristoyl-2-palmityl phosphatidylcholine; 1-palmityl-2-myristoyl phosphatidylcholine; 1-palmityl-2-stearyl phosphatidylcholine; 1-stearyl-2-palmityl phosphatidylcholine; dioleyl phosphatidyl choline; the dioleate PHOSPHATIDYL ETHANOLAMINE; two Laurel acyl phospholipids acyl glycerol; phosphatidylcholine; Phosphatidylserine; PHOSPHATIDYL ETHANOLAMINE; phosphatidylinositols; sphingomyelins; sphingolipid; phosphatidyl glycerol; diphosphatidylglycerol; two myristoyl phosphatidyl glycerols; DPPG; DSPG; the dioleoyl phosphatidyl glycerol; Dimyristoyl phosphatidic acid; two palmityl phosphatidic acid; two myristoyl PHOSPHATIDYL ETHANOLAMINE; two palmityl PHOSPHATIDYL ETHANOLAMINE; two myristoyl Phosphatidylserine; DPPS; cephalin acyl serine; the brain sphingomyelins; two palmityl sphingomyelins; distearyl vaginula phospholipid; phosphatidic acid; galactocerebroside; ganglioside; cerebroside; the dilauryl phosphatidylcholine; (1; 3)-D-mannose acyl group-(1,3) two glyceride; the aminobenzene glycosides; 3-cholesteryl-6 '-(glycosyl sulfo-) hexyl ether glycolipid and cholesterol and derivant thereof.
Be applicable to peptide of the present invention including but not limited at 3 common pending applications [in JIUYUE in 1997 application on the 29th, series number _ _, proxy number is 9196-0004-999,9196-0005-999,9196-0006-999] the middle kind of putting down in writing, it all is introduced in this as a reference.
Although be not all essential in each case, preferably should or remove the precipitate dissolving before mixing or stirring lipid and peptide solution or before the lyophilization.
The method can be used for mass preparation peptide/peptide/lipid complex formation, both sexes peptide/(phosphorus) peptide/lipid complex formation, contaminated with lipid protein/(phosphorus) peptide/lipid complex formation and/or ApoAl peptide analogues/(phosphorus) peptide/lipid complex formation.Can prepare the lyophilization raw material that is used for the large volume preparation, perhaps, will be before lyophilization blended peptide/lipid solution be placed in (as unit dose package) in the smaller container, these less packings can be made aseptic single dose dosage form.
The present invention can provide the vacuum drying compositions of single dose or multiple-unit container form, method is that the sterile solution before the vacuum drying is divided in the suitable container to the capacity of stipulating, prepare required vacuum drying compositions, then with single dose or multi-dose container sealing.By dried composition in situ hydration being reconstituted the suitable sterile solution with required administration concentration, thereby in the container of embedding, form solution fast with suitable sterile diluent." suitable container " used herein refers to keep gnotobasis, can adopt the plug seal mode to carry the container of vacuum drying product, as vial.In addition, suitable container has suitable size, is suitable for the volume of the solution after the vacuum drying compositions hydration reconstruct; With the suitable containers material, be generally I class glass.Used stopper, for example aseptic rubber stopper or same device should be able to provide above-mentioned sealing, and adding diluent-as the inlet of Injectable sterile water (USP), normal saline (USP) or 5% D/W (USP), to reconstitute required solution also can be arranged.Container container as described in the present application is known to these and other aspect of the suitability of medicine to the technical staff of pharmaceutical field.In specific embodiments, the unit dose of product can be 10mg to a 2g peptide, and preferred 100mg to 1g reconstitutes concentration behind the solution and be 1 to 50mg/ml, preferred 2-25mg/ml.
Method of the present invention makes the preparation of protein or peptide/peptide/lipid complex formation can be to animal or human's parenteral, comprise vein, intraperitoneal, subcutaneous, intramuscular injection and as the contrast agent micelle intravenous injection of diagnosis usefulness, or to the animal or human by oral, rectum, mucosa delivery (as oral mucosa) or topical, or be used for experiment in vitro.
The lyophilization powder of the inventive method preparation can reconstitute solution at once before injection, or selects directly to take the lyophilization powder.The lyophilization powder comprises the lipid and the peptide of the complex that can form microcapsule, liposome, sphere or forms such as disc granule, micelle granule, but is not limited to this.For making the lyophilization powder reconstitute solution or rehydrated, select solution according to required final use.Select sterile solution when being used for medicine.And then, for the preferred buffer solution of specific purposes, comprise phosphate, citrate, Tris, baribital, acetate, glycine-HCl, succinate, cacodylate, boric acid-Borax, ammediol and carbonate.
Can prepare lyophilization powder of the present invention with any Freeze Drying Technique known in the art, comprise lyophilization, but not only be confined to use lyophilization the freezing back of the solution that contains peptide/lipid reduction vaporization.
This method also is applicable to and is stored in unstable or undissolved chemical compound when lacking lipid.
This method can be used to prepare the product of treatment or prevention human diseases, comprise application as common antigen-presenting in vaccine, treatment or prevention dyslipoproteinemia-lack including, but not limited to hypercholesterolemia, hypertriglyceridemia, low HDL and aPoA-I, reach cardiovascular disease, septicemia or infectious disease such as arteriosclerosis.
The method can also prepare the complex as pharmaceutical carrier, as the vehicle (transmission medicine, DNA, gene) of transmitting medicine as cell outside liver or liver, or as the cleanser of catching toxin (as insecticide, LPS etc.).Perhaps, this method also can prepare the complex that is used for extracorporeal diagnostic system or shadowgraph technique.
In specific embodiments, can prepare the complex of ApoA-I analog (including, but not limited to its agonist) with this method, be used for the in-vitro diagnosis method and as the mark of DHL crowd and subgroup.In other specific embodiments, ApoA-I agonist complex can be used for immunoassay or shadowgraph technique (as cat scan, MRT scanning).
Following example is in order to explanation the present invention, but all can not think limitation of the present invention in either side.
6. embodiment: prepare peptide-peptide/lipid complex formation with freeze-drying altogether
Prepare peptide lipid complex in order to following scheme.
With peptide 1 (PVLDLFRELLNELLEALKQKLK; SEQ ID NO:1) (22.4mg) is dissolved in insulation several minutes and rotation mixing discontinuously in the methanol, makes the solution of 3.5mg/ml.The methanol solution (100mg/ml storing solution) that adds two palmityl phosphatidylcholines (DPPC) in this solution makes that the final ratio of DPPC/ peptide is 2.5: 1 (W/W) in the solution.This solution rotating is mixed.Adding dimethylbenzene to final concentration in this solution is 36%.Take out part gained solution and carry out analysed by gel filtration chromatography.In liquid nitrogen that solution is freezing, and vacuum freezing is to doing.The part (0.9%NaCl) in Sterile Saline that will contain 20mg peptide 1 (SEQ ID NO:1) and 50mgDPPC is rehydrated, mixes and is heated to 41 ℃, keeps a few minutes, until the peptide/phospholipid complex settled solution that produces reconstruct.
6.1 example: use gel filtration and phospholipid
6.1.1 raw material and method
For the preparation condition of test complex, often get used to preparing a spot of complex and identify.These preparations contain the 1mg peptide, prepare as follows: in the cleaning glass bottle of a 1.0ml tool plug (Waters#WAT025054), 1mg peptide 1 (SEQID NO:1) is dissolved in 250 other methanol of μ l HPLC level (Perkin Elmer).Rotate bottle at ambient temperature frequently to impel the peptide dissolving.After 10 minutes, can also see a small amount of undissolved granular substance, but harmless for result of the test.From DPPC concentration be take out respectively the methanol solution storing solution of 100mg/ml contain 1,2,3,4,5,7.5,10 or several parts of test solutions of 15mg DPPC (Avanti polarity lipid, purity 99%, production number #850355) add in the mixture.Add methanol and make the volume of mixture reach 400 μ l, further be interrupted the rotation mixture 10 minutes in room temperature.Almost can not see not molten material at Guan Zhongyi this moment.In every pipe, add 200 μ l dimethylbenzene (Sigma-Aldrich purity 99%, HPLC rank) and rotated 10 seconds.Open 2 duck eyes at the top of every pipe with No. 20 standard syringe syringe needles, with test tube in liquid nitrogen freezing 15 seconds, test tube is lyophilized overnight under vacuum condition.In every pipe, add 200ml 0.9%NaCl solution.With every test tube rotation 20 seconds.Solution appearance in the test tube is emulsus at this moment.Test tube is incubated 30 minutes at 41 ℃.Except that the test tube that contains 15mgDPPC still is that the solution in all test tubes all becomes clarification (being that outer appearnce is similar to water) the emulsus.
For measure institute whether be useful on the phospholipid of preparation complex all actual appear at corresponding to the post eluting of chromatograph absworption peak partly in, by the post eluate that the volume of every part of 1ml or 2ml is collected reconstruct peptide/peptide/lipid complex formation, the content of enzymatic assays phospholipid is described according to manufacturer with BioMerieux phospholipid Enzymatique PAP150 test kit (#61491).
Also can the large-scale production complex.More than reported the example of this preparation.These mixture are used in vivo test.
6.2 the qualification result of complex
Fig. 1: Superose 6 chromatograms of the ripe HDL that from 200 μ l human serums, prepares with the density supercentrifugation.Chromatogram is presented at the absorption of 254nm.Elution volume=14.8ml, being equivalent to Stokes ' diameter is 108 dusts (seeing Table 1).
Fig. 2 (end): in above-mentioned (on a small scale preparation) insulation ratio (Ri is defined as the ratio of total phospholipids and total peptide in original mixture) is 1: 1 (w: w) Zhi Bei DPPC: Superose 6 chromatograms of peptide 1 complex.The retention volume of absworption peak is 16.2ml and 18.1ml, and being equivalent to the Stokes ' diameter littler than HDL is the granule of 74 and 82 dusts.Comprise the eluting of absworption peak partially recycled 87% (seeing Table 1) of upper prop phospholipid.
Fig. 2 (top): in above-mentioned Ri=2: 1 (w: the DPPC of ratio preparation w): Superose 6 chromatograms of peptide 1 complex.The retention volume of absworption peak is 16.4ml, is equivalent to the granule littler than HDL (77 dusts).Comprise the eluting of absworption peak partially recycled 70% (seeing Table 1) of upper prop phospholipid.
Fig. 3 (end): in above-mentioned Ri=3: 1 (w: the DPPC of ratio preparation w): Superose 6 chromatograms of peptide 1 complex.The retention volume of absworption peak is 16.0ml, is equivalent to the granule littler than HDL (80 dusts).Comprise the eluting of absworption peak partially recycled 79% (seeing Table 1) of upper prop phospholipid.
Fig. 3 (top): in above-mentioned Ri=4: 1 (w: the DPPC of ratio preparation w): Superose 6 chromatograms of peptide 1 complex.The retention volume of absworption peak is 15.7ml, is equivalent to the granule littler than HDL (90 dusts).Comprise the eluting of absworption peak partially recycled 106% (seeing Table 1) of upper prop phospholipid.
Fig. 4 (end): in above-mentioned Ri=5: 1 (w: the DPPC of ratio preparation w): Superose 6 chromatograms of peptide 1 complex.The retention volume of absworption peak is 15.1ml, is equivalent to the granule littler than HDL (104 dusts).Comprise the eluting of absworption peak partially recycled 103% (seeing Table 1) of upper prop phospholipid.
Fig. 4 (top): in above-mentioned Ri=7.5: 1 (w: the DPPC of ratio preparation w): Superose 6 chromatograms of peptide 1 complex.The retention volume of absworption peak is 13.6ml, is equivalent to the granule bigger than HDL (134 dusts).Comprise the eluting of absworption peak partially recycled 92% (seeing Table 1) of upper prop phospholipid.
Fig. 5: in above-mentioned Ri=10: 1 (w: the DPPC of ratio preparation w): Superose 6 chromatograms of peptide 1 complex.The retention volume of absworption peak is 13.4ml, is equivalent to the granule bigger than HDL (138 dusts) equally.Comprise the eluting of absworption peak partially recycled 103% (seeing Table 1) of upper prop phospholipid.
Owing to contain DPPC: the ratio of peptide 1 is the sample muddiness of 15: 1 complex, does not therefore handle with Superose 6 chromatographic columns, and this also points out is that big granule is arranged.
Each above-mentioned test except that containing the eluting part of the eluate corresponding with absworption peak, does not detect significant phospholipid (seeing Fig. 2-8) in other parts.In fact this pointed out the complex enclose all phospholipid (in the test error of method).Test shows by changing the initial proportion of phospholipid and peptide, can obtain (bigger or little than HDL) even complex of various volumes.6.3 use 14C-labelling peptide 1 is identified complex
Contain with the preparation of above-mentioned freeze-drying altogether 14Peptide-phospholipid the complex of C-labelling peptide 1 (specific activity is by weight 159, and the 000DMP/mg peptide is supposed and contained 50% peptide).Every kind of preparation contains 1mg peptide and 3,4 or 5mg DPPC.Complex after the reconstruct, is handled 20 μ l (100 μ g) complex in 200 μ l 0.9%NaCl with Pharmacia Superose 6 posts, as liquid phase, flow velocity is 0.5ml/min with 0.9%NaCl.(the blank volume of post=7.7ml), collect the flow point of 1ml behind the delay volume of 5ml.With the sample that contains 20 μ l flow points, with the BioMerieux enzymatic assays content of phospholipid wherein.The remainder of every part of flow point places Wallach 1410 liquid scintillation counters (Pharmacia), counts 3 minutes with quick counting procedure (the Easy Count program).Analysis result is shown in Fig. 6-8.As seen, respectively the peak volume be about 16,16 and the minority flow point of 15ml in, reclaim the DPPC that has obtained in 3: 1,4: 1 and 5: 1 simultaneously: the phospholipid and the peptide of the overwhelming majority in the complex of peptide ratio preparation.The UV absorption curve of these samples shows: by DPPC: the ratio of peptide is 3: 1,4: 1 and the complex for preparing at 5: 1, respectively 15.1,14.7 and the volume place of 14.4ml eluting (dead volume that fraction collector and UV detect between the flow cell is 1.3ml, and what this can be interpreted as and have little difference between the elution volume that radioactivity/phospholipid determination method and UV absorption process record) from the post.Ri is that the elution volume of 3: 1,4: 1,5: 1 complex corresponds respectively to 106,114 and 120 dust Stoke ' s diameters.
Table 1
DPPC: peptide 1 ratio Elution volume Particulate relative volume The % of the phospholipid that in absworption peak, occurs
????HDL ????14．8 ????- ????-
????1∶1 16.2 and 18.1 Little ????87％
????2∶1 ????16．4 Little ????70％
????3∶1 ????16．0 Little ????79％
????4∶1 ????15．7 Little ????106％
????5∶1 ????15．1 Little ????103％
????7．5∶1 ????13．6 Greatly ????92％
????10∶1 ????13．4 Greatly ????103％
????15∶1 ????ND ????ND ????ND
Compare ¨ ND with the particulate volume of HDL, do not do
The invention is not restricted to the described scope of specific embodiments, only as the single explanation to various aspects of the present invention, method that function is identical and composition are also within the scope of the invention for they.In fact, drawn with accompanying drawing from the above description, except that record of the present invention to various improvement of the present invention, to it will be readily apparent to those skilled in the art that.These improvement will fall in the protection domain of appended claims.

Claims (37)

1. method for preparing cryodesiccated peptide/lipid product, comprise will be in a solvent system one or more can take the peptide or the peptide analogues of both sexes configuration, be total to lyophilization with one or more lipid and form peptide/lipid product, wherein said product can rehydrated formation peptide/peptide/lipid complex formation.
2. method according to claim 1, wherein said peptide are contaminated with lipid protein.
3. method according to claim 1, wherein said peptide analogues are the analog of ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, Apo E or other apoprotein.
4. method according to claim 1, wherein said peptide is a protein.
5. method according to claim 1, wherein said lipid are natural lipid, synthetic lipid, saturated lipid, unsaturated lipid or its mixture.
6. method according to claim 5; wherein said lipid is selected from following material: the lecithin phatidylcholine; the fabaceous lecithin phatidylcholine; ether phospholipid (ether phospholipid); lower alkyl chains phospholipid; cholesterol; cholesterol derivative; two palmityl phosphatidylcholines; L-Dimyristoylphosphatidylcholine; DSPC; 1-myristoyl-2-palmityl phosphatidylcholine; 1-palmityl-2-myristoyl phosphatidylcholine; 1-palmityl-2-stearyl phosphatidylcholine; 1-stearyl-2-palmityl phosphatidylcholine; dioleyl phosphatidyl choline; the dioleate PHOSPHATIDYL ETHANOLAMINE; two Laurel acyl phospholipids acyl glycerol; phosphatidylcholine; Phosphatidylserine; PHOSPHATIDYL ETHANOLAMINE; phosphatidylinositols; sphingomyelins; sphingolipid; phosphatidyl glycerol; diphosphatidylglycerol; two myristoyl phosphatidyl glycerols; DPPG; DSPG; the dioleoyl phosphatidyl glycerol; Dimyristoyl phosphatidic acid; two palmityl phosphatidic acid; two myristoyl PHOSPHATIDYL ETHANOLAMINE; two palmityl PHOSPHATIDYL ETHANOLAMINE; two myristoyl Phosphatidylserine; DPPS; cephalin acyl serine; the brain sphingomyelins; two palmityl sphingomyelins; distearyl vaginula phospholipid; phosphatidic acid; galactocerebroside; ganglioside; cerebroside; the dilauryl phosphatidylcholine; (1; 3)-D-mannose group-(1,3) two glyceride; the aminobenzene glycosides; 3-cholesteryl-6 '-(glycosyl sulfo-) hexyl ether glycolipid and composition thereof.
7. method according to claim 1, also be included in vacuum lyophilization before, in the process or afterwards with the step of product sterilization.
8. method according to claim 1, wherein said peptide/peptide/lipid complex formation is aseptic.
9. method according to claim 1 also is included in before the vacuum lyophilization described peptide and described lipid branch is packed in the independent container, to form the dosage form of unit dose.
10. the pharmaceutical dosage form of a unit dose wherein contains the aseptic cryodesiccated peptide/lipid mixtures by the method preparation of claim 1 or 7.
11. method for preparing lyophilization peptide/lipid product, comprise: (a) analog with at least a both sexes peptide or peptide is dissolved in first solution, (b) at least a lipid is dissolved in second solution, wherein said second solution and first solution are miscible, (c) described first solution and described second solution are mixed, form peptide/lipid solution, (d) with described peptide/lipid solution lyophilization, to generate cryodesiccated peptide/lipid product, it can rehydrated formation peptide/peptide/lipid complex formation.
12. the described method of claim 11, wherein said peptide are contaminated with lipid protein.
13. the described method of claim 1, wherein said peptide is a protein.
14. method according to claim 9, wherein said peptide analogues are the analog of ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, Apo E or other apoprotein.
15. method according to claim 11, wherein said lipid are natural lipid, synthetic lipid, saturated lipid, unsaturated lipid or its mixture.
16. method according to claim 15; wherein said lipid is selected from following material: the lecithin phatidylcholine; cholesterol; cholesterol derivative; ether phospholipid (ether phospholipid); the fabaceous lecithin phatidylcholine; lower alkyl chains phospholipid; two palmityl phosphatidylcholines; L-Dimyristoylphosphatidylcholine; DSPC; 1-myristoyl-2-palmityl phosphatidylcholine; 1-palmityl-2-myristoyl phosphatidylcholine; 1-palmityl-2-stearyl phosphatidylcholine; 1-stearyl-2-palmityl phosphatidylcholine; dioleyl phosphatidyl choline; the dioleate PHOSPHATIDYL ETHANOLAMINE; two Laurel acyl phospholipids acyl glycerol; phosphatidylcholine; Phosphatidylserine; PHOSPHATIDYL ETHANOLAMINE; phosphatidylinositols; sphingomyelins; sphingolipid; phosphatidyl glycerol; diphosphatidylglycerol; two myristoyl phosphatidyl glycerols; DPPG; DSPG; the dioleoyl phosphatidyl glycerol; Dimyristoyl phosphatidic acid; two palmityl phosphatidic acid; two myristoyl PHOSPHATIDYL ETHANOLAMINE; two palmityl PHOSPHATIDYL ETHANOLAMINE; two myristoyl Phosphatidylserine; DPPS; cephalin acyl serine; the brain sphingomyelins; two palmityl sphingomyelins; distearyl vaginula phospholipid; phosphatidic acid; galactocerebroside; ganglioside; cerebroside; the dilauryl phosphatidylcholine; (1; 3)-D-mannose group-(1,3) two glyceride; the aminobenzene glycosides; 3-cholesteryl-6 '-(glycosyl sulfo-) hexyl ether glycolipid and composition thereof.
17. method according to claim 11, wherein said peptide/lipid solution is aseptic.
18. method according to claim 11, wherein said peptide/peptide/lipid complex formation is aseptic.
19. method according to claim 11 also is included in before the vacuum lyophilization in the independent container that described peptide/lipid solution branch packed into, to form the dosage form of unit dose.
20. method according to claim 11, also be included in vacuum lyophilization before, in the process or afterwards with the step of product sterilization.
21. the pharmaceutical dosage form of a unit dose, it contains the aseptic stable cryodesiccated peptide/lipid mixtures by the method preparation of claim 11 or 19.
22. peptide/peptide/lipid complex formation with following method preparation, its method comprises: to form the peptide/lipid product of dehydration, this product can rehydrated generation peptide/peptide/lipid complex formation with one or more both sexes peptide in the solvent system or peptide analogues and at least a lipid vacuum lyophilization.
23. the described peptide/peptide/lipid complex formation of claim 22, wherein said peptide are contaminated with lipid protein.
24. the described peptide/peptide/lipid complex formation of claim 22, wherein said peptide are the ApoAl analog.
25. the described peptide/peptide/lipid complex formation of claim 22, wherein said lipid are natural, synthetic, saturated, unsaturated lipid or its mixture.
26. peptide/peptide/lipid complex formation according to claim 25; wherein said lipid is selected from following material: the lecithin phatidylcholine; the fabaceous lecithin phatidylcholine; cholesterol; cholesterol derivative; lower alkyl chains phospholipid; ether phospholipid; two palmityl phosphatidylcholines; L-Dimyristoylphosphatidylcholine; DSPC; 1-myristoyl-2-palmityl phosphatidylcholine; 1-palmityl-2-myristoyl phosphatidylcholine; 1-palmityl-2-stearyl phosphatidylcholine; 1-stearyl-2-palmityl phosphatidylcholine; dioleyl phosphatidyl choline; the dioleate PHOSPHATIDYL ETHANOLAMINE; two Laurel acyl phospholipids acyl glycerol; phosphatidylcholine; Phosphatidylserine; PHOSPHATIDYL ETHANOLAMINE; phosphatidylinositols; sphingomyelins; sphingolipid; phosphatidyl glycerol; diphosphatidylglycerol; two myristoyl phosphatidyl glycerols; DPPG; DSPG; the dioleoyl phosphatidyl glycerol; Dimyristoyl phosphatidic acid; two palmityl phosphatidic acid; two myristoyl PHOSPHATIDYL ETHANOLAMINE; two palmityl PHOSPHATIDYL ETHANOLAMINE; two myristoyl Phosphatidylserine; DPPS; cephalin acyl serine; the brain sphingomyelins; two palmityl sphingomyelins; distearyl vaginula phospholipid; phosphatidic acid; galactocerebroside; ganglioside; cerebroside; the dilauryl phosphatidylcholine; (1; 3)-D-mannose group-(1,3) two glyceride; the aminobenzene glycosides; 3-cholesteryl-6 '-(glycosyl sulfo-) hexyl ether glycolipid and composition thereof.
27. the described peptide/peptide/lipid complex formation of claim 22, wherein said complex is aseptic.
28. the described peptide/peptide/lipid complex formation of claim 22 wherein is mixed with aseptic unit dose with described complex.
29. aseptic, a cryodesiccated compositions wherein contains the sterile preparation of the complex that analog and lipid by the peptide that can take the both sexes alpha-helical conformation or peptide forms.
30. preparation according to claim 28, wherein said preparation are aseptic single dose dosage form.
31. a cryodesiccated compositions, it contains peptide/peptide/lipid complex formation, and peptide wherein is the analog of contaminated with lipid protein or ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III I, Apo E or other apoprotein.
32. the freeze-dried composition of claim 31, peptide/peptide/lipid complex formation wherein are microcapsule, micelle, liposome, discoid granule, spherical particle or its mixture.
33. a freeze-dried composition that contains peptide/peptide/lipid complex formation, peptide wherein can be taked the both sexes alpha-helical conformation.
34. the described freeze-dried composition of claim 33, peptide/peptide/lipid complex formation wherein are microcapsule, micelle, liposome, discoid granule, spherical particle or its mixture.
35. claim 31 or 33 described compositionss, wherein said compositions is aseptic.
36. claim 29 or 31 described compositionss, wherein said analog is not peptide or protein.
37. method for preparing lyophilization peptide/lipid product, comprise: in the solvent system one or more can be taked the peptide of both sexes alpha-helical conformation or the analog of peptide, with one or more lipid, in peptide is the ratio of about 2-200 than lipid, competent time of lyophilization altogether, with form can be in solution the peptide/lipid product of rehydrated generation peptide/peptide/lipid complex formation.
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